The plasmid-encoded type Ill secretion system of Yersinia pestis functions to deliver a set of toxins (Yops) into host eukaryotic cells. The Yop toxins modulate signaling pathways in host cells to neutralize innate immune mechanisms. The toxin YopJ triggers apoptosis in macrophages infected with Yersinia. YopJ is structurally related to a family of cysteine proteases, but the precise target(s) of its toxic activity remain unknown. It has been shown that YopJ inhibits several signaling pathways that are responsible for activation of transcription factors. The signaling pathway that activates the transcription factor NF-kB is a key target of YopJ. It is hypothesized that YopJ promotes macrophage death by reducing expression of one or more apoptosis inhibitor genes that are regulated by NF-kB. We will use microarray analysis to determine if any genes encoding apoptosis inhibitors are expressed at lower levels in macrophages infected with wild-type Y. pestis as compared to macrophages infected with YopJ Y. pestis. Apoptosis inhibitor genes that are down regulated in a YopJ-specific manner will be overexpressed in macrophages to determine if their products can protect against Yersinia-induced cell death. The experiments proposed in this R21 application will augment specific aim 3 of the parent R01 grant (AI43389-03 "Modulation of Host Signaling Functions by Yersinia Yops"). Specific aim 3 is to elucidate the mechanism of YopJ-induced apoptosis by identifying functional interactions between YopJ and components of host signaling pathways. The proposed experiments are consistent with the exploratory/developmental nature of the R21 application because they will employ established genomic approaches to characterize genome-wide transcriptional responses and to facilitate gene discovery.